HIGH-RESOLUTION STRUCTURE OF THE PHOSPHORYLATED FORM OF THE HISTIDINE-CONTAINING PHOSPHOCARRIER PROTEIN HPR FROM ESCHERICHIA-COLI DETERMINED BY RESTRAINED MOLECULAR-DYNAMICS FROM NMR-NOE DATA

The solution structure of the phosphorylated form of the histidine-con taining phosphocarrier protein, HPr, from Escherichia coli has been de termined by NMR in combination with restrained molecular dynamics simu lations. The structure of phospho-HPr (P-HPr) results from a molecular dynamics simulation in water, using time-dependent distance restraint s to attain agreement with the measured NOEs. Experimental restraints were identified from both three-dimensional H-1-H-1-N-15 HSQC-NOESY an d two-dimensional H-1-(1)HNOESY spectra, and compared with those of th e unphosphorylated form. Structural changes upon phosphorylation of HP r are Limited to the active site, as evidenced by changes in chemical shifts, in (3)J(NHH alpha)-coupling constants and NOE patterns. Chemic al shift changes were obtained mainly for protons that were positioned close to the phosphoryl group attached to the Hisl5 imidazole ring. D ifferences could be detected in the intensity of the NOEs involving th e side-chain protons of Hisl5 and Pro18, resulting from a change in th e relative position of the two rings. In addition, a small change coul d be detected in the three-bond T-coupling between the amide proton an d the H-alpha proton of Thr16 and Arg17 upon phosphorylation, in agree ment with the changes of the phi torsion angle of these two residues o btained from time-averaged restrained molecular dynamics simulations i n water. The proposed role of the torsion-angle strain at residue 16 i n the mechanism of Streptococcus faecalis HPr is not supported by thes e results. In contrast, phosphorylation seems to introduce torsion ang le strain at residue Hisl5. This strain could facilitate the transfer of the phosphoryl group to the A-domain at enzyme II. The phospho-hist idine is not stabilised by hydrogen bends to the side-chain group of A rgl7; instead stable hydrogen bonds are formed between the phosphate g roup and the backbone amide.protons of ThrlG and Argl7, which show the largest changes in chemical shift up on phosphorylation, and a hydrog en bond involving the side-chain O-y proton of Thr16. HPr accepts the phosphoryl group from enzyme I and donates it subsequently to the A do main of various enzyme II species. The binding site for EI on HPr rese mbles that of the A domain of the mannitol-specific enzyme II, as canb e concludedfrom the changes onthe amideprotonandnitrogenchemical shif ts observed via heteromolecular single-quantum coherence spectroscopy.